The incidence of obesity and type 2 diabetes has reached global epidemic prevalence in both the developed and developing countries. Patients with glucocorticoid (GC) excess (Cushing's syndrome) develop reversal obesity and insulin resistance. However, patients with idiopathic obesity and/or metabolic syndrome do not have elevated circulating GC levels. Metabolic tissues such as liver and adipose tissue solely express 112- hydroxysteroid dehydrogenase (112-HSD1) where it acts in intact cells and organs as a NADPH-dependent reductase to generate active cortisol from inert circulating cortisone. 112-HSD1 therefore plays a crucial role in local steroid reactivation. In the endoplasmic reticulum (ER) lumen, hexose-6-phosphate dehydrogenase (H6PDH) converts glucose-6-phosphate (G6P) and NADP to generate NADPH. This reaction requires the G6P transporter (G6PT) to maintain its metabolic substrateG6P availability. This generates NADPH which is utilized by 112-HSD1, thus constituting a connection of H6PDH and 112-HSD1 in regulation of tissue GC regeneration. In support of this, H6PDH knockout mice are unable to regenerate tissue GCs and impaired 112- HSD1 activity. We recently reported that reduction of H6PDH expression leads to suppressed hepatic and adipose 112-HSD1 activity that may contribution to insulin sensitivity and weight loss in a high- fat diet model of obesity. Our new preliminary data show that tissue-specific activation of H6PDH expression leads to up- regulation of 112-HSD1 activity that correlated with insulin resistance and obesity in type 2 diabetic animal models. We therefore hypothesize that H6PDH is the critical determinant of whether 112-HSD1 can regenerate GCs that contributes to the pathogenesis of type 2 diabetes and obesity. In this grant, we will examine the impact of H6PDH on insulin signaling action through manipulation of H6PDH to regulation of 12-HSD1 amplifying GC regeneration by using siRNA technology in intact mouse hepatocytes and adipocytes. These cell culture studies will be reinforced by treatment of animals with specific H6PDH or G6PT siRNA in a viral vector to define that inhibition of H6PDH could provide therapeutic benefits for diabetes and obesity. We believe that blocking the effects of H6PDH on 112-HSD1 may be a new strategy in the effective treatment of diabetes/or obesity Public Health Relevance: Patients who produce too much cortisol develop obesity and in some cases, type 2 diabetes. We posit this is due, in part, to the H6PDH-induced 112-HSD1 amplifying tissue glucocorticoid (GC) regeneration. Blocking the effects of H6PDH on 112-HSD1 may represent a new strategy in the effective treatment of type 2 diabetes and obesity through reduction of tissue GC availability mediating insulin sensitivity and glucose homeostasis.